Combined multidimensional single-cell protein and RNA profiling dissects the cellular and functional heterogeneity of thymic epithelial cells

The network of thymic stromal cells provides essential niches with unique molecular cues controlling T cell development and selection. Recent single-cell RNA sequencing studies have uncovered previously unappreciated transcriptional heterogeneity among thymic epithelial cells (TEC). However, there are only very few cell markers that allow a comparable phenotypic identification of TEC. Here, using massively parallel flow cytometry and machine learning, we deconvoluted known TEC phenotypes into novel subpopulations. Using CITEseq, these phenotypes were related to corresponding TEC subtypes defined by the cells’ RNA profiles. This approach allowed the phenotypic identification of perinatal cTEC and their physical localisation within the cortical stromal scaffold. In addition, we demonstrate the dynamic change in the frequency of perinatal cTEC in response to developing thymocytes and reveal their exceptional efficiency in positive selection. Collectively, our study identifies markers that allow for an unprecedented dissection of the thymus stromal complexity, as well as physical isolation of TEC populations and assignment of specific functions to individual TEC subtypes.


Field-specific reporting
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Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. n/a n/a n/a n/a Sample size was chosen based on pilot experiments and previous experience with similar experiments to achieve sufficient statistical power. Data were collected from two or more biological replicates.
No data was excluded from analysis.
All experiments were repeated and reliably reproduced. Data were collected from three biological replicates if not stated differently. Number of independent experiments performed are indicated in the figure legends.
No randomization of mice was performed. Litter mates, where applicable, or age-and gender-matched mice were used as controls.
Blinding was not undertaken as only wild-type animals at distinct ages were analyzed with the exception of data shown in Figure 4f,g and Supplementary Figure 10 were gene targeted animals were specifically and knowingly used to verify findings. The methodological approaches used, massively parallel flow cytometry and machine learning , did not explicitly allow for blinding. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots. Antibodies were validated and quality tested for flow cytometry as stated by the manufacturers. If required the antibody concentrations used were further optimized by titration prior to use.
The study did not involve wild animals.
Both male and female animals were used. Gender-matched wild-type mice were used in all experiments as a reference for genetically modified animals.
The study did not involve samples collected from the field.
Animals were maintained under specific pathogen-free conditions at the University of Oxford Biomedical Science facilities. Experiments were performed according to institutional and U.K. Home Office regulations and age-and gender-matched wild-type mice were used in all experiments as a reference for genetically modified animals. Rag2-/-mice were bred and maintained in the mouse facility of the Department of Biomedicine at the University of Basel in accordance with permissions and regulations of the Cantonal Veterinary Office of Basel-Stadt.
Isolated thymi were cleaned from adipose tissue, separated into the two lobes, and subsequently subjected to three rounds of enzymatic digestion with Liberase (2.5 mg/ml, Roche) and DNaseI (10 mg/ml, Roche) diluted in PBS (Sigma) at 37°C. After filtration through a 100 m cell strainer and resuspension in FACS buffer (PBS supplemented with 2% FBS (Sigma)), cell number was determined using a CASY cell counter (Innovatis). For most analyses CD45+ hematopoietic cells were depleted by incubation with anti-CD45 beads (Miltenyi) as per manufacturer`s recommendations and subsequently subjected to the AutoMACS separator (Miltenyi) "depleteS" program. Cells were counted and stained in FACS buffer containing antibodies of interest for 30 min at 4°C in the dark. For intra-cellular staining, cells were fixed and permeabilised after cell-surface staining using the Cytofix/Cytoperm (BD Biosciences) or the Fix/Perm buffer set (Invitrogen) according to the manufacturer`s protocol.
BD LSRFortessa and BD FACSAria III were used for analysis and sorting.